红椿SSR-PCR体系建立和多态性引物筛选

Establishment and Primer Screening of SSR-PCR Reaction System for Toona ciliata

[目的]本研究旨在建立红椿SSR-PCR最佳反应体系,并筛选适于红椿SSR分析的高多态性引物。[方法]通过L16(45)正交试验设计,确立红椿SSR-PCR最佳反应体系;利用优化后的体系对来自楝科植物的135对SSR引物,在6个不同的红椿居群中进行扩增,筛出能有效扩增的引物并进一步筛选出适于红椿的高多态性引物。[结果](1)10μL基于荧光d UTP的SSR-PCR体系中包含:10×buffer 1.0μL,Taq酶(5 U·μL-1)0.1μL,Mg Cl2(25mmol·L-1)0.8μL,d NTP(200 mmol·L-1)0.025μL,荧光d UTP(1 nmol·μL-1)0.01μL,引物(10 mmol·L-1)0.8μL,DNA模板45 ng剩余用dd H2O补足;(2)筛选出29对能有效扩增的引物,复选后获得了12对适于红椿SSR分析的高多态性引物。[结论]建立了SSR-PCR最佳反应体系并筛选出高多态性引物,为红椿的分子标记等遗传学研究提供了基础。

Objective]To establish SSR-PCR reaction system,and screen high polymorphism primers for SSR anal-ysis of Toona ciliata.[Method]The orthogonal design was employed to obtain the optimal SSR-PCR system of T. ciliate.Using optimized system of SSR-PCR,135 pairs of SSR primers from Meliaceae plants were amplificated in six populations.[Result]The optimal 10 μL reaction system contained 1.5 μL 30 ng·μL -1 Template DNA,0.8μL 10 umol·L -1 primers,0.025 μL 10 mol·L -1 dNTPs,0.8 μL 25mol·L -1 Mg2 ＋,0.1 μL 5 U·μL -1 taq DNA polymerase,0.01μL 1 mol·L -1 F-dUTP,1μL 10 × PCR buffer （Mg2 ＋free）and 5.765 μL ddH2 O.12 primers with high polymorphism were selected among 29 primer combinations.[Conclusion]The optimal reaction system of SSR-PCR were established and high polymorphism primers were selected,which could be used in analysis of fingerprinting genetic diversity,molecular breeding and variety identification in T.ciliate.